Cell culture and transfection
Human melanoma cells (A875 and SK-MEL-110) were obtained from the Cell Bank of the Chinese Academy of Sciences. All cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Using NOD2 (shNOD2-1 ,5′-GGGCAAGACTTCCAGGAATTT-3′; shNOD2-2, 5′- GTGCTTCTTTGCCGCGTTCTA-3′; shNOD2-3, 5′-GGACTACAACTCTGTGGGTGA-3′) and its control (CON313) and NOD2 overexpression and its control (CON335) lentiviruses infected A875 and SK-MEL-110 cells. These viruses were obtained from GeneChem Co., Ltd. (Shanghai, China). After 48 hours of transfection, the cell transfection efficiency was observed under a fluorescence microscope, and the cells were continuously screened by puromycin (Solarbio, #P8230) pressurization until the infection rate reached more than 95%. Real-time PCR and Western blot assessed the effect of gene transfection.
Real-time PCR
TRIzol reagent (Takara, #9109) was used to extract total RNA from cells and tumor tissues. Synthesize cDNA by reverse transcription of mRNA following the instructions of the Reverse Transcription Kit (Thermo Scientific, #K1622). cDNA was analyzed by Real-time PCR using SYBR Green Master mix (Roche, #04913914001); U6 served as an internal reference gene. The specific primer sequences are detailed in Table 1.
Table 1
Gene | primer |
NOD2 | F: 5’- 3’: TGGTTCAGCCTCTCACGATGA R: 5’- 3’: CAGGACACTCTCGAAGCCTT |
TYMS | F: 5’- 3’: CTGCTGACAACCAAACGTGTG R: 5’- 3’: GCATCCCAGATTTTCACTCCCTT |
PLK1 | F: 5’- 3’: AAGTGGGTGGACTATTCG R: 5’- 3’: GCCGTCACGCTCTATGTA |
Cyclin E1 | F: 5’- 3’: ACTCAACGTGCAAGCCTCG R: 5’- 3’: GCTCAAGAAAGTGCTGATCCC |
CDK2 | F: 5’- 3’: CCAGGAGTTACTTCTATGCCTGA R: 5’- 3’: TTCATCCAGGGGAGGTACAAC |
Cyclin D1 | F: 5’- 3’: GCTGCGAAGTGGAAACCATC R: 5’- 3’: CCTCCTTCTGCACACATTTGAA |
CDK4 | F: 5’- 3’: TCAGCCAGCTTGACTGTTCCA R: 5’- 3’: GCCTAGATTTCCTTCATGCCA |
P16 | F: 5’- 3’: GGGTTTTCGTGGTTCACATCC R: 5’- 3’: CTAGACGCTGGCTCCTCAGTA |
Bax | F: 5’- 3’: CCCGAGAGGTCTTTTTCCGAG R: 5’- 3’: CCAGCCCATGATGGTTCTGAT |
Bcl2 | F: 5’- 3’: GGTGGGGTCATGTGTGTGG R: 5’- 3’: CGGTTCAGGTACTCAGTCATCC |
Caspase 3 | F: 5’- 3’: CATGGAAGCGAATCAATGGACT R: 5’- 3’: CTGTACCAGACCGAGATGTCA |
U6 | F: 5’- 3’:CTCGCTTCGGCAGCACA R: 5’- 3’: AACGCTTCACGAATTTGCGT |
Western blot
Cells and tissues can be effectively lysed using a RIPA lysis buffer (Solarbio, #R0020) containing protease and phosphatase (Solarbio, #P0100) inhibitors. Proteins were quantified by BCA, separated via SDS-PAGE gel electrophoresis, and transferred onto PVDF membranes (Millipore, #IPVH00010), sealed with 5% skimmed milk or 5% BSA (Bio Froxx, #4240GR500) for two hours then incubated overnight with a primary antibody at 4°C. The next day, the secondary antibody of the same genus was used and incubated at room temperature for two hours. ELC chemiluminescence was performed to observe the target bands, and ImageJ software was used to scan the gray values.
The following antibodies were used: anti-NOD2 (Abcam, #ab36836), anti-TYMS (Proteintech, #15047-1-AP), anti-PLK1 (Santa Cruz, #sc-17783), anti-p-PLK1(T210) (Abcam, #ab155095), anti-Cyclin E1 (Proteintech, #11554-1-AP), anti-Cyclin D1 (Proteintech, #60186-1-Ig), anti-CDK2 (Proteintech, #10122-1-AP), anti-CDK4 (Proteintech, #11026-1-AP), anti-P27 (Proteintech, #25614-1-AP), anti-Bcl2 (Proteintech, #68103-1-Ig), anti-Bax (Proteintech, #60267-1-Ig), anti-Caspase 3 (Proteintech, #66470-2-Ig), anti-MMP2 (Proteintech, #10373-2-AP), anti-MMP9 (Proteintech, #10375-2-AP), anti-N-cadherin (Proteintech, #22018-1-AP), anti-E-Cadherin (Proteintech, #20874-1-AP), anti-vimentin ( Proteintech, #60330-1-Ig), anti-Ubiquitin (CST, #43124), anti- Myc-tag (MCE, HY-P80232), anti- GST-Tag (MCE, HY-P80148), anti-β-actin (Proteintech, #66009-1-Ig), anti-rabbit IgG (CST, #7074), and anti-mouse IgG (Santa Cruz, #sc2005).
Cell proliferation assay (MTS, colony formation, and EdU staining)
To determine cell viability by MTS assay, cells with NOD2 overexpression and knockdown of A875 and SK-MEL-110 were cultured in 96-well plates (1000 cells/well). MTS (Promega, #CTB169) working solution formulated according to 1:5 (DMEM: MTS) was added at different time points. The absorbance value was detected at 490 nm after one hour of dark culture.
Cells were inoculated into six-well plates (800 cells/well), and fresh medium was replaced every three days. Discontinue the culture when cell colony formation was observed. Cells were washed with PBS, fixed in paraformaldehyde, stained with 0.5% crystal violet for 20 minutes each, and photographed for counting.
In the EdU incorporation determination, cells were first grown in 24-well plates, and the next day, an EdU Kit (Beyotime, # C0078S) was used to stain the cells. Hoechst 33342 was utilized for nuclear staining. Pictures were obtained using a fluorescence microscope (Leica, # DM4B) to determine the percentage of EdU-positive cells.
IC50 assay
Cells were inoculated in 96-well plates at 5000 cells/well density. The next day, cells were exposed to various fluorouracil (5-FU) (MCE, #HY-90006) and capecitabine (CAP) (MCE, #HY-B0016) treatment concentrations. The IC50 was calculated by detecting the absorbance values at 490 nm obtained in the MTS assay described above after 48-hour exposure.
Wound healing assay
Cells were inoculated into a six-well plate and incubated to reach confluence after overnight incubation with serum-free DMEM. Further, the cells were scratched with the tip of a 200-uL pipette, and the separated cells were washed using PBS. Images were taken under an inverted microscope at 0 and 24 hours after wounding. Calculate the wound closure area as follows: wound closure area (fold) = (initial wound area - unhealed wound area 24 hours after scratching)/initial wound area.
Transwell migration and invasion assays
Cells were digested with trypsin, suspended in DMEM, and later cultured into the top Chamber in Transwell chambers (Corning, #3524). When performing Transwell invasion experiments, 40 µl of matrix gel (BD, #356234) diluted 1:3 (matrix gel: DMEM) was applied to the upper chamber of the Transwell chamber two hours in advance. Next, 600 uL of medium containing 10% fetal bovine serum was injected into the lower chamber of the Transwell, and cells that had migrated and invaded the bottom of the Transwell were removed after 24 hours. Finally, the migrated and invaded cells were fixed, crystal violet-stained, and counted.
Flow cytometry
Cell cycle assays are performed by inoculating cells into a 6-well plate, digesting, and centrifuging. Then, they were slowly added dropwise to 75% pre-cooled ethanol and fixed at 4°C for 24 hours. Cells were collected by centrifugation the next day, stained with a cycle kit (4A Biotech, #FXP0211), and subjected to flow cytometry detection (BD, BD FACSCCelestaTM flow cytometry) and analyzed using FlowJo software.
In apoptosis analysis, cells were inoculated in 6-well plates and treated for apoptosis induction. After staining with Annexin V, 633/PI Staining Kit (Dojindo, #AD11) according to the manufacturer's instructions, assessed apoptic content through flow cytometry (BD, BD FACS Celesta TM flow cytometer) and analyzed using FlowJo software.
Co-immunoprecipitation (CO-IP)
The cells were lysed for 30 minutes on a shaker at 4°C using weak RIPA lysis buffer (Beyotime, #P0013D), adding protease and protease phosphatase (Solarbio, #P0100) inhibitors. Following centrifugation, the supernatant was divided into three parts. The input tube was frozen at minus 20°C, and the corresponding primary antibody was mixed in the IgG and IP tubes. After incubation on a shaking table at low temperatures for 8 hours, protein A/G-Agarose beads (Roche, #11243233001) were added and gently shaken at 4°C overnight. After centrifugation, the liquid above the sediment was removed, and the sample was subsequently rinsed thrice with PBS. Finally, PBS and protein loading buffer were added, followed by boiling and eluting the combined protein. Western blot analysis was performed using SDS-PAGE.
Cellular immunofluorescence (IF)
After inoculating the cells in a slide culture, they were removed and fixed in 4% paraformaldehyde for 15 min, followed by exposure to 0.3% Triton X-100 for 20 min. Next, blocking with BSA (Bio Froxx, #4240GR500) for 2 hours, followed by the addition of the primary antibody for overnight incubation at 4°C. Samples with a fluorescent secondary antibody were incubated in the dark for one hour, and the slide was sealed with an anti-fluorescence quencher containing DAPI (Sigma, #F6057). Finally, the cell slides were photographed using a fluorescence microscope (Zeiss, #LSM800) observation. The following fluorescent secondary antibodies were utilized: 488 (Proteintech, #SA00013-1) and 594 (Proteintech, #SA00013-4).
GST Pull-down assay
The GST-TYMS and myc-PLK1 genes were first cloned and synthesized and then inserted into the pGEX-4T-1 vector and pET28a (+) vector, respectively, and then expressed individually in E. coli TOP10, which was purchased from Wuhan Jinkai Rui Biological. For the in vitro pull-down assay, performed according to the illustrated procedure (Fitgene, #FI88807), after the bacteria were broken by ultrasonication in lysis buffer, the protein samples of 2 mg of GST (control) or GST-TYMS (experimental) were incubated with 50 µl of glutathione agarose resin for 5 h at 4°C. The samples were washed with rinse buffer three times and then washed with rinsing buffer. After washing three times with rinse buffer, 2 mg of myc-PLK1 protein was mixed into each control and experimental group and incubated at 4°C overnight. Then, the samples from both groups were centrifuged, washed three times with rinse solution, and eluted by adding elution buffer for 15 min. Add protein loading buffer to the eluted proteins and heat at 95°C for 10 minutes. Finally, the samples were analyzed by immunoblotting.
TYMS activity assay
Total proteins were extracted with RIPA lysis buffer to determine intracellular TYMS enzyme activity, followed by concentration determination using the BCA method. Following guidelines provided by the manufacturer, TYMS activity was assayed using the TYMS Activity ELISA Kit (MEIMIAN, #MM-0328H1).
Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) analysis
Animal tumors were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5-µm sections, and stored after baking at 65°C for three hours. HE staining was performed per the manufacturer's established protocol (Solarbio, #G1120).
For IHC, antigen repair was performed under high pressure using citrate buffer. The endogenous peroxidase activity was inactivated with 3% H2O2, and 5% BSA was blocked, followed by the addition of the primary antibody and overnight incubation at 4°C. Staining Sections using the DAB Substrate Kit (Biosharp, #BL732A) and hematoxylin staining solution (Biosharp, #517-28-2). Sections were dehydrated again, cleared by a gradient of ethanol and xylene, sealed with neutral glue, and photographed under the microscope (Teksqray, #SQS-1000).
Bioinformatics analysis
Data mining
Mining was conducted for data normalization, and computational analysis was performed of the NOD2 gene expression values obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo) for the mRNA expression dataset GSE15605, as well as for normal and primary melanoma tissues and metastatic melanoma tissues in the TCGA database (https://portal.gdc.cancer.gov/). The GEPIA database (http://gepia2.cancer-pku.cn/) was used for the differential analysis of the NOD2, TYMS, and PLK1 genes in melanoma and normal tissues, as well as the correlation between TYMS and PLK1 in melanoma patients. The UALCAN database (https://ualcan.path.uab.edu/) was used for survival prognosis analysis of NOD2 expression and melanoma patients.
For transcriptome sequencing (RNA-seq), SK-MEL-110 cells with NOD2 overexpression and control were collected for RNA-seq by Zhongke New Life in Shanghai, China. After selecting the high-throughput sequencing data according to the screening threshold of | log2FC | > 1 and Padj < 0.05, differentially expressed genes (DEGs) were obtained and enriched for analysis.
Animal models
Six-week-old female BALB / c nude mice were obtained from the Department of Laboratory Animals, Kunming Medical University, and all animal experiments followed animal ethical standards. For the no administration of chemotherapeutic drugs treatment group, 1 × 107 of A875-shCAD and its control cells were implanted into the subcutaneous regions on both sides of the nude mice., respectively. After two weeks, measure tumor diameters with a vernier caliper every 5 days. For the group treated with chemotherapeutic agents, 1 × 107 A875 cells were implanted into the axilla of nude mice. After two weeks, the nude mice were injected intraperitoneally with saline or chemotherapeutic drugs 5-Fu (25 mg/kg) and BI6727 (10 mg/kg) every three days, and the diameter of the tumors was measured using vernier calipers every five days the treatment period. The tumor volume was calculated using the formula: volume = 1/2 × long diameter × wide diameter × wide diameter. In the final stage of the tumor experiment, we euthanized the nude mice and removed tumors for subsequent experiments.
Statistical analysis
Data were analyzed using GraphPad Prism 2 software. All data were shown as means ± standard deviation (SD) of at least three independent experiments. Independent-sample t-tests were used to compare differences between two specific groups, and one-way analysis of variance (ANOVA) was used to compare differences between multiple groups. P < 0.05 was considered to indicate statistically significant differences in the results.